The main hindrance to develop antiviral therapies and effective vaccines for HIV/AIDS is the high variability of the virus. Recombination is a major mechanism that is responsible for this rapid diversification of HIV-1 population. My current research focuses on the mechanisms and restrictions in HIV-1 intersubtype recombination. We have shown that the dimerization initiation signal (DIS) sequence is a major determinant of intersubtype recombination and that the DIS has an important function in maintaining dimeric RNA structure important for recombination. To further understand the role of recombination in HIV-1 evolution, we investigate how recombination contributes to the generation of recombinants with altered replication fitness. Our long-term goal is to elucidate the elements in the viral genome that affect replication fitness of a HIV-1 recombinant. These viral elements represent new potential targets for blocking or enhancing recombination that can affect the continuous replication of HIV-1 in the host. The objectives of this application are to reveal 1) the proportion of recombinants generated in cell culture and patients that are viable, 2) the ratios at which these recombinants will have better, equal and worse replication fitness compared to wildtype and 3) the elements in viral genome that determine replication fitness. The central hypothesis is that newly generated HIV-1 intersubtype recombinants have an array of replication fitness and there is a finite window for HIV-1 replication fitness allowing the recombinants to continue to replicate in the host. This hypothesis is based on the observations that CRF02_AG has a higher replication fitness than the parental subtypes A and G HIV-1 and that our preliminary data demonstrated recombination between subtypes B and C HIV-1 can generate recombinant with altered replication fitness. The rationale of the proposed research is that characterizing the replication fitness of HIV-1 intersubtype recombinants will allow us to further understand the molecular mechanisms for generating new HIV-1 recombinant strains with biological advantages. The Specific Aims are to i) determine the replication fitness of cell culture-derived HIV-1 intersubtype recombinants, ii) identify the location of crossover junctions of patient-derived HIV-1 intersubtype recombinants, and iii) characterize the replication fitness of patient-derived HIV-1 intersubtype recombinants. The proposed research is relevant to public health because the identified viral elements that determine replication fitness may represent new targets for developing strategies to prevent the continuous replication of HIV-1 in the human hosts.